Goal of this work is to understand why pancreatic ?-cells fail with an eye toward identifying genetic, biochemical, and cellular pathways that can be exploited asdrug targets to prevent and reverse this disease process. During its ten-year life, this grant has suppoted original observations identifying a homeostatic loop orchestrated by transcription factor FoxO1 tha integrates hormonal and nutrient signals into a gene expression program to preserve ?-cell function and lineage stability. Signal achievements of the last funding cycle have been the demonstration tht ?-cell failure, long held to be secondary to loss of ?-cells to apoptosis, is due to ?-cell dediffeentiation into endocrine progenitor-like cells that express Neurogenin3, and their partial conversion into oter hormone-producing cells. In preliminary data presented to support a continuation of these studies, he PI shows that mice lacking FoxO1, 3a, and 4 in pancreatic progenitors or differentiated ?-cells devlop MODY, with striking abnormalities of the transcriptional networks regulated by Hnf4a, Hnf1a, and Pd1. Transcriptome analyses of the different mutants suggest a model in which FoxO control metabolic flexibility in differentiated ?-cells, and acquisition of glucose competence in maturing ?-cells. Te PI seeks continuing support for this work in three interrelated aims. In Aim 1 the PI will investigatethe reversibility and prevention of ?-cell dedifferentiation, using genetic, pharmacological, and developmental tools. To this end, the PI will use a robust model of insulin-resistant diabetes, speifically developed to aid this work. In preliminary data to support Aim 2, the PI shows that triple knockoutof Foxo1, 3a, and 4 in ?-cells impair insulin secretion, results in mitochondrial dysfunction, impaire formation and trafficking of intracellular vesicles, reduced Ca signaling and branched chain amino cid metabolism, and increased lipid oxidation. The PI proposes to test the hypothesis that FoxO regulat the ?-cell's metabolic flexibility, i.e. the ability to select between glucose and non-glucose secretagogues as energy sources for mitochondrial ATP synthesis required for insulin secretion. In im 3, comparative transcriptome analyses of FoxO-deficient pancreatic progenitors vs. differentiated ? cells reveal that genes required for glucose uptake and metabolism are affected in the former, but ot in the latter. The PI hypothesizes that this gene set is specifically regulated by FoxO in maturing ?-ells, and bestows on ?-cells glucose competence, i.e. the ability to properly sense glucose that is requied for ?-cell maturation. The PI proposes to use a combination of developmental and epigenetic analyse coupled with candidate gene approaches to understand the mechanism by which FoxO control ?-cell maturation.